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Molecular dissection of HERV-W dependent microglial- and astroglial cell polarization

ABSTRACT: The endogenous retrovirus type W (HERV-W) is a human specific entity, which was initially discovered in multiple sclerosis (MS) patient derived cells. We initially found that the HERV-W envelope (ENV) protein negatively affects oligodendrogenesis and controls microglial cell polarization towards a myelinated axon associated and damaging phenotype. Such first functional assessments were conducted ex vivo, given the human specific origin of HERV-W. Recent experimental evidence gathered on a novel transgenic mouse model, mimicking activation and expression of the HERV-W ENV protein, revealed that all glial cell types are impacted and that cellular fates, differentiation, and functions were changed. In order to identify a HERV-W specific signature in glial cells, the current study analyzed the transcriptome of ENV stimulated microglial- and astroglial cells and compared the transcriptomic signature to lipopolysaccharide (LPS) stimulated cells, owing to the fact that both ligands can activate toll-like receptor-4 (TLR-4). Additionally, a comparison between published disease associated glial signatures and the transcriptome of HERV-W ENV stimulated glial cells was conducted. We, therefore, provide here for the first time a detailed molecular description of specific HERV-W ENV evoked effects on those glial cell populations that are involved in smoldering neuroinflammatory processes relevant for progression of neurodegenerative diseases.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE262029 | GEO | 2024/03/30

REPOSITORIES: GEO

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Project description:Human Endogenous Retroviruses (HERVs) are exogenous viral elements that integrated into the germline millions of years ago. They comprise 8% of the human genome and play a critical role in cellular differentiation during development. HERV-K(HML-2) is the most recently integrated and actively transcribed HERV. It is found to be highly expressed in pluripotent stem cells and strongly downregulated during cellular differentiation. Moreover, expression of HERV-K in terminally differentiated neurons results in cytotoxicity. Expression of HERVs has been implicated in carcinogenesis and is suggested to confer stem cell like features important for cell growth. Atypical Teratoid / Rhabdoid Tumor (AT/RT) is a rare pediatric brain cancer that results from incomplete neuronal differentiation during embryonic development. In this paper, we investigated the cytopathic effect of Ouabain, a cardiac glycoside that induces cytotoxicity in stem cells, on AT/RT cells. We characterized four AT/RT cell lines by immunostaining and observed variable levels of stem cell, Oct4 and Pax6, and neuronal differentiation markers, TUBB3 and MAP2, as well as HERV-K envelope (env), polymerase (pol), and gag transcripts. We stained formalin-fixed brain tissues from 20 AT/RT patients and 5 unaffected controls for HERV-K ENV protein expression. 40% of AT/RT patient tissues showed ENV expression. In contrast, no ENV immunostaining was observed in all 5 control brain tissues. We used Ouabain, known to be cytotoxic to stem cells, observed up to 50% toxicity at 24 hr and 80% toxicity at 48 hr after treatment. While AT/RT primarily exist as large cell aggregates in suspension, Ouabain treatment downregulated HERV-K ENV protein and caused disruption of cell-to-cell interactions. Similarly, when AT/RT cells were transfected with a CRISPR/Cas9 construct designed to repress HERV-K promotor activity, cell aggregates separated and cell viability decreased significantly. Using a luciferase reporter construct under the control of a HERV-K promotor, we observed a significant decrease in HERV-K promotor activity at 8 to 72 hr post treatment with 0.5 uM Ouabain. Given that downregulation of HERV-K alone was sufficient to cause cytotoxicity in the cells, HERV-K expression may be essential for AT/RT growth and survival. Ouabain and other compounds that modulate HERV expression may provide an alternative or adjunctive therapeutic option for treatment of embryonal tumors.

Project description:To check the acetylation levels at the positions harbouring HERV-loci and assess their variation after the treatment with HDACis (Vorinostat, romidepsin), the univocal genomic coordinates of about 3280 HERV-loci have been compared with the position of the H3AcK9 peaks identified by ChIP-seq analysis. The first analysis confirmed that HDACis are able to modulate the acetylation of repetitive elements, including HERV sequences. Subsequently, we considered only the differential peaks (diffpeaks), i.e. the peaks showing significant variation after the treatment with vorinostat and/or romidepsin. Interestingly, even if more than half of HERV-loci co-localised with acetylation peaks after HDACi treatment, only a minority of them (0.9 to 1.9%) co-localised with diffpeaks, showing mostly a reduction in their acetylation levels. Diffpeaks co-localised HERV-loci could be classified into 24 HERV groups and included the ERV-V2 locus. We focused then on the 11 HERV-loci which co-localised with diffpeaks showing an increase in acetylation levels, hence indicating an increased transcriptional activation after HDACi treatment. Remarkably, diffpeaks corresponding to the ERV-V2 locus were identified in all samples and were the most significantly upregulated in both vorinostat and romidepsin treatment. This was in part confirmed by ChIP-qPCR in different OC cells. Beside ERV-V2, only the HML8 sequence at locus 2q11,2 showed increased acetylation levels in all treated samples, independent from the HDACis used, but having lower statistical significance. The analysis revealed that the treatment with HDACis did not only account for the significant increase of ERV-V2 acetylation, but also led to the shift and concentration of acetylation peaks at the 5’ region of the HERV provirus. This observation allows us to hypothesize that HDACi treatment could not only increase ERV-V2 transcriptional activation, but also modify its pattern of expression, and eventually leading to alternative splicing.

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Molecular dissection of HERV-W dependent microglial- and astroglial cell polarization

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